Project 2: Summary/Abstract Acute pancreatitis, an inflammatory disease of the pancreas, causes death in over 30% of those with severe disease. Specific therapies to prevent pancreatitis or reduce injury are lacking. The disease usually originates in the pancreatic acinar cell after exposure to specific insults that cause pathologic acinar cell signaling. These changes in acinar cell signaling change the function of specific target proteins and cause organelle dysfunction. Among the most characteristic acinar cell early pancreatitis responses are intracellular activation of digestive enzymes (especially proteases) and inhibited secretion. Recent studies from Program investigators have also shown that there is decreased lysosomal function in acute pancreatitis. This Project will examine two recently characterized proteins (AP3 and D52) that can modulate zymogen activation, secretion, and may also affect autophagy and lysosomal function. We hypothesize that these two proteins regulate the biogenesis and trafficking of the minor secretory compartment (MSC), a pathway needed to form zymogen granules (ZG) from immature secretory granules (ISG). We posit that this compartment also regulates apical secretion and contributes to the normal function of the endo-lysosomal pathway. Finally, we propose a new model for zymogen activation at the onset of pancreatitis that begins with zymogen activation in the MSC then proceeds to decreased degradation of active enzymes by lysosomes. Three Specific Aims relating to AP3 and D52 and its binding partner, Rab5, are planned: 1) Investigate their role in regulating physiologic secretion and membrane trafficking 2) Examine their expression and localization in pancreatitis models 3) Determine whether changing their expression levels affects pancreatitis responses. Finally, we will collaborate with other Projects to determine whether these molecules affect other pathways under study. Studies are planned using isolated mouse and human pancreatic acinar cells and in vivo mouse pancreatitis models. Our preliminary studies indicate that during pancreatitis, the levels of expression of these proteins change. Further, over-expression or deletion of these proteins by genetic or molecular means has dramatic effects on lysosomal protein trafficking, zymogen activation, and cell injury. Preliminary findings also suggest that the functions of AP3 and D52 may interact with pathways under study by other projects, such as Rab GTPases in Project 1, autophagic pathways in Project 3, and ER stress in Project 4. Extensive use of the Program's Animal and Pathology, Vector and Human Acinar, and Resource Management and Biostatistics Cores is planned.